The human body contains a million or so distinct proteins. Chemistry harbors the potential to provide ready access to these natural proteins as well as to create nonnatural ones with desirable attributes. During the previous grant period, new chemical means were discovered to manipulate protein structure and protein function. In particular, the "traceless Staudinger ligation" was developed for the synthesis of peptide and other amide bonds. This chemoselective reaction between a phosphinothioester and azide proceeds rapidly and in high yield, and leaves no residual atoms in the product. Specific Aims. The overall goal of the proposed research is to use ideas and methods from organic chemistry and chemical biology to extend our fundamental understanding of the chemical reactivity of proteins, and to employ that understanding in meaningful ways. During the next grant period, this intent will be achieved in five Specific Aims. Aim 1 is to explore the utility of a new chemical reaction, the phosphinoester-mediated reductive fragmentation of azides into diazo-compounds. Aim 2 is to use light as a means to control the traceless Staudinger ligation on a micron scale. Aim 3 is to use the traceless Staudinger ligation to synthesize otherwise inaccessible ubiquitin conjugates so as to reveal imperatives of ubiquitin-mediated protein degradation. Aim 4 is to develop olefin metathesis as an orthogonal reaction of high utility for protein chemistry. Finally, Aim 5 is to develop peptidic and small-molecule catalysts for oxidative protein folding. Significance. The results of the research proposed herein will provide new insight into the intrinsic and extrinsic chemical reactivity of proteins, as well as extend the capacity to access and manipulate proteins. The knowledge gained will have a broad and deep impact on biomedicine in this post-genomic era. PUBLIC HEALTH RELEVANCE: Most genes encode proteins. A protein is a string of amino acids that folds into a three-dimensional structure. Proteins perform the molecular functions that are necessary for life. These functions include catalysis of biochemical reactions (by enzymes), neutralization of foreign toxins (by antibodies), and stimulation of cellular activity (by hormones). The goal of this project is to develop chemical means both to gain access to proteins and to manipulate proteins with a precision that is unobtainable with other (e.g., genetic) methods.